Our long-term goal is to determine the molecular mechanism for the photochemical events in visual excitation. We want to understand how rhodopsin shifts the absorption maximum of its 11-cis retinal protonated Schiff base chromophore from 440 nm to 500 nm in rod pigments, and how this interaction is altered in blue and red cone pigments. The mechanism of the 11-cis yield 11-trans photoisomerization and of energy storage in the primary photoproduct, bathorhodopsin, will also be determined. These goals will be addressed by using resonance Raman scattering to obtain vibrational spectra of the protein bound chromophore. Vibrational analyses will then be used to determine chromophore structure. The specific aims are: 1) Resonance Raman spectra will be obtained of rhodopsin, isorhodopsin and bathorhodopsin pigments whose retinal chromophores have been labeled with 13C and 2H. Isotopic derivatives will also be used to assign the vibrations of the 11-cis, 9-cis and all-trans retinal Schiff base model compounds. Comparison of these frequencies and assignments will allow us to determine the "opsin shift" of the C-C and C=C modes. These empirical opsin shifts should allow us to pinpoint the location(s) of the opsin perturbations responsible for Lambda max-regulation and energy storage. 2) Vibrational force field calculations will be performed and protein-induced changes in the force constants, normal modes, and Raman intensities will be analyzed to provide more quantitative information on the nature of these protein perturbations. 3) Magic angle sample spinning 13C-NMR spectra will be obtained of rhodopsin and isorhodopsin regenerated with specific 13C-labeled retinals. The chemical shifts, relaxation times, and tensor elements will be used as an additional probe of chromophore structure and chromophore-protein interactions. 4) Models for chromophore structure and protein-chromophore interactions in rhodopsin and bathorhodopsin will be developed and evaluated with QCFF-Pi calculations. 5) Resonance Raman microscopy will be used to study the mechanism(s) of Lambda max-regulation in a variety of rod and cone photoreceptors to test the generality of the ideas developed in (1)-(4). 6) In a related project, resonance Raman spectra of retinoid binding proteins will be examined to determine the structure of the bound retinal chromophore. This work will tell us how these proteins solubilize retinoids for transport to and from the retinal rod cell, and should provide additional information on how protein-chromophore interactions produce opsin shifts.